Anthracycline antibiotics FA-1180 B and B.sub.1, or salts thereof

Abstract

An anthracycline antibiotic FA-1180 A, A.sub.1, B or B.sub.1, or salts thereof, said antibiotic having the formula (I): ##STR1## wherein R represents an acetyl group or a 1-hydroxyethyl group.

Description

BACKGROUND OF THE INVENTION

This invention relates to novel anthracycline antibiotics FA-1180 A, A.sub.1, B and B.sub.1, or salts thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are each an optical micrograph of the spore chains of strain FA-1180;

FIGS. 3, 4 and 5 are each an electron micrograph of the spore chains of strain FA-1180;

FIG. 6 is another electron micrograph of the spore chains of strain FA-1180;

FIG. 7 is an IR spectrum of antibiotic FA-1180 A;

FIG. 8 is an IR spectrum of antibiotic FA-1180 A.sub.1 ;

FIG. 9 is an IR spectrum of antibiotic FA-1180 B;

FIG. 10 is an IR spectrum of antibiotic FA-1180 B.sub.1 ;

FIG. 11 is a UV and visible spectrum of antibiotic FA-1180 A;

FIG. 12 is a UV and visible spectrum of antibiotic FA-1180 A.sub.1 ;

FIG. 13 is a UV and visible spectrum of antibiotic FA-1180 B;

FIG. 14 is a UV and visible spectrum of antibiotic FA-1180 B.sub.1 ; and

FIG. 15 is a PMR spectrum of antibiotic FA-1180 A.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that the new strain of actinomycetes, FA-1180 strain isolated from the soil collected on the shore of Lake Biwa, Shiga, Japan, has the ability to produce a novel antibiotic FA-1180 A, A.sub.1, B or B.sub.1 (hereunder referred to as antibiotic A, A.sub.1, B or B.sub.1). The antibiotic has strong ability to inhibit the growth of Gram-positive microorganisms, and it also has antitumor activity. Therefore, it is useful as an active ingredient of a medicine such as a disinfectant for medical devices or chemotherapeutics to treat bacteriasis in human beings or animals.

The antibiotic of this invention can be produced, for example, by extracting, separating and purifying from the culture broth of the strain FA-1180. The strain used in such fermentation is a novel actinomycetes that was isolated from the soil on the shore of Lake Biwa, designated Actinomadura roseoviolacea var. biwakoensis nov. var. (hereunder referred to as the Strain) and deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, as FERM-P 5155, and its mycological properties are as follows.

(1) Morphological properties:

For observation of its morphological properties, the Strain was cultivated on seven media, yeast extract-malt extract agar medium (ISP No. 2), oatmeal agar medium (ISP No. 3), ISP No. 3 medium containing B vitamins (ISP No. 3V), inorganic salts-starch agar medium (ISP No. 4), ISP No. 4 medium containing B vitamins (ISP No. 4V), glycerol-asparagine agar medium (ISP No. 5), and ISP No. 5 medium containing B vitamins (ISP No. 5V).

No fragmentation or separation of substrate mycelium was observed in either medium. Aerial mycelium was not observed in ISP No. 4 or ISP No. 5, slightly observed in ISP No. 5V, and a long straight or flexible aerial mycelium was observed in ISP No. 2, ISP No. 3, ISP No. 3V and ISP No. 4V. Spore chains were formed on the tips of very short aerial mycelia which were, generally, monopodially branched from the long aerial mycelium, and they looked like globular sporangia (3 to 8.mu. in size) under an optical microscope (see FIGS. 1 and 2 which are each an optical micrograph of the spore chains of strain FA-1180). Such morphological features were observed in ISP No. 3, ISP No. 3V and ISP No. 4V, but little in ISP No. 2 and ISP No. 5V. Direct observation with an electron microscope or indirect observation by carbon replication revealed that the spore chains formed a spiral wound closely two to five turns into a doughnut (FIGS. 3, 4 and 5 which are each an electron micrograph of the spore chains of strain FA-1180), but spore chains that resembled a pseudosporangium were also found (FIG. 6 which is another electron micrograph of the spore chains of strain FA-1180). The spores were oval, 0.8.about.1.1.times.0.6.about.1.0.mu. in size and had a smooth surface.

ISP No. 3V, ISP No. 4V and ISP No. 5V were prepared by adding the following B vitamins to one liter of ISP No. 3, ISP No. 4 and ISP No. 5: 0.5 mg of thiamine hydrochloride, 0.5 mg of riboflavin, 0.5 mg of niacin, 0.5 mg of pyridoxine hydrochloride, 0.5 mg of inositol, 0.5 mg of calcium pantothenate, 0.5 mg of p-aminobenzoic acid and 0.25 mg of biotin.

(2) Cultural characteristics of Actinomadura roseoviolacea var. biwakoensis nov. var. on various media.

TABLE 1__________________________________________________________________________Cultural Characteristics of Actinomaduraroseoviolacea var. biwakoensis nov. var. on Various MediaMedium andAbbreviation Thereof Growth Aerial Mycelium Soluble Pigment__________________________________________________________________________A Trypton-yeast Fair, Pale yellowish Restricted, Slightly, extract broth flaky growth on bottom White.about.pale Bright reddish (ISP No. 1) of tube, Partially pink orange light orange.about.strong reddish orange ring on surface, in contact with glassB Yeast extract-malt Good, Remarkably Fair, Slightly, extract agar raised, Many folds, Pink.about.purplish Strong reddish (ISP No. 2) Strong orange.about.deep pink orange reddish orange, turning later into deep redC Oatmeal agar Fair.about.somewhat Fair, Strong reddish (ISP No. 3) and restricted, Flat, Powdery, Some orange ISP No. 3V Spreading, Strong drops of color- reddish orange less guttation, White.about.pale pinkD Inorganic salts- Restricted, Penetrat- Scant, White None starch agar ing into medium, Light (ISP No. 4) yellowish brownE ISP No. 4V Good, Somewhat raised, Fair.about.good, Strong orange Strong reddish orange Pale pink zone around growthF Glycerol-asparagine Very poor, Light None None agar (ISP No. 5) yellowish brownG ISP No. 5V Good, Remarkably Restricted, Slightly, raised, Strong reddish White.about.pale Bright orange orange.about.deep reddish pink orangeH Peptone-yeast Moderate, Raised, Many None Slightly, extract-iron agar fine folds, Grayish Deep red (ISP No. 6) brown.about.dark grayish brownI Tyrosine agar Fair.about.somewhat None None (ISP No. 7) restricted, Flat, Dull red, turning later into deep red purpleJ ISP No. 7V Fair, Flat, Deep red None Deep red purple purpleK Sucrose nitrate agar Fair, Light orange.about. Restricted, None or (Waksman No. 1) strong reddish orange White.about.pale slightly, pink Reddish orangeL Waksman No. 1V Fair.about.good, Bright Fair, Pale Deep red purple orange.about.deep red pink.about.pale purple yellowish pinkM Glucose-asparagine Poor, Pale yellow None None agar (Waksman No. 2)N Waksman No. 2V Fair.about.good, Somewhat Scant, White.about. Bright orange raised, Bright reddish pale pink orangeO Bouillon agar Fair, Dull red.about. Very scant, Dull red (Waksman No. 8) grayish red WhiteP Glucose-bouillon Very good, Remarkably Very scant, Dull red agar raised, Many folds, White Dull red.about.dark redQ Potato scab Fair, Somewhat raised, Restricted, Grayish pink.about. (Waksman No. 40) Strong reddish orange.about. White.about.grayish grayish red dull purplish red pinkR Loffler's blood Fair.about.somewhat None Slightly, Deep serum restricted, Bright red purple yellowish red, turning later into deep red purpleS Skim milk Fair.about.somewhat None Slightly, Light (Waksman No. 41) restricted, Ring, orange.about.bright Bright orange.about.bright orange reddish orangeT Calcium malate agar Very poor or no growth None NoneU Calcium malate Somwhat restricted, None Vivid red agar V Flat, Spreading, purple Vivid red purpleV Glycerol-calcium Restricted.about.poor, None None malate agar Bright orange (Waksman No. 7)W Waksman No. 7V Fair, Dull red None None or slight- ly dull redX Bouillon-gelatin No growth stab (20.degree. C.)__________________________________________________________________________ The media identified by V in the column of "Media and their abbr." of Table 1 were prepared in the same manner as in the preparation of the media containing B vitamins, described before.

The respective tests were conducted in accordance with the methods described in International Journal of Systematic Bacteriology, Vol. 16, p. 313, in 1966 and S. A. Waksman, The Actinomycetes, Vol. 2. Unless otherwise noted, all media were held at 27.degree. to 28.degree. C. for 21 days for incubation of the Strain, and color observation was made in compliance with Manual of Color Names, published by Nippon Shikisai Kenkyu-jo in 1973.

After the 21-day incubation on ISP No. 2, ISP No. 3, ISP No. 3V, ISP No. 4, ISP No. 4V, ISP No. 5 and ISP No. 5V, the Strain was treated with 0.05 N hydrochloric acid and 0.05 N sodium hydroxide, and the results indicated in Table 2 below were obtained.

TABLE 2______________________________________Change in Growth and Soluble Pigment with pH Growth 0.05N or 0.05N No SodiumMedia Soluble Hydrochloric Treat- Hydroxide(abbr.) Pigment Acid Treatment ment Treatment______________________________________1 ISP No. 2 Growth Dull red Dark Deep red purplish red Soluble Dull yellowish Dull Dull red pigment orange orange purple2 ISP No. 3 Growth No change Bright Light and (bright orange grayish ISP No. 3V orange) purple Soluble Bright reddish Bright Bright pigment orange orange purple.about. light grayish purple3 ISP No. 4V Growth No change Vivid Bright (vivid reddish reddish purple orange) orange4 ISP No. 5V Growth No change Bright Bright (bright orange purple orange)______________________________________

(3) Physiological characteristics

(a) Growth temperature range (tested on a yeast extract-malt extract broth every five degrees in the range of from 15.degree. to 50.degree. C.): Grew up to 40.degree. C.

Optimum temperature range: 35.degree. to 40.degree. C.

(b) Liquefaction of gelatin: negative

(c) Peptonization of skim milk: positive (weak and slow)

Coagulation of skim milk: positive (weak and slow)

(d) Liquefaction of Loffler's blood serum: negative

(e) Production of melanoid pigment:

Trypton-yeast extract broth: negative Tyrosine agar medium: negative Peptone-yeast extract-iron agar medium: negative

(f) H.sub.2 S formation: negative

(g) NH.sub.3 formation: negative

(h) Nitrate reduction: positive (strong)

(i) Starch hydrolysis: positive (weak)

(4) Utilization of carbon sources (tested on ISP No. 9 medium)

TABLE 3______________________________________Utilization of Various Carbon SourcesGrowth Carbon Sources______________________________________Positive D-Glucose, L-Arabinose, D-Galactose,(good or D-Fructose, Salicin, Sucrose, L-Rhamnose,fair growth) Inositol, D-Xylose, D-Mannose, D-Mannitol, Maltose, D-Lactose, Trehalose, Cellobiose, MelibioseDoubtful D-SorbitolNo Inulin, Raffinose(Addition of B vitamins promoted the growth.)______________________________________

(5) Cell wall composition

The Strain was cultured in a trypton-yeast extract agar medium (ISP No. 1) and a purified wall was prepared in accordance with the method of T. Yamaguchi described in J. Bacteriol., Vol. 89, pp. 444-453, 1965, and subjected to an analysis. The wall was subjected to amino acid analysis by a Yanagiomoto High-Speed Amino Acid Analyzer. Stereoisomers of .alpha.,.epsilon.-diaminopimelic acid were determined by the method of Hoare et al., described in Biochem. J., Vol. 65, pp. 441-447, 1957. Sugar determination was made by a GLC mass spectral analysis (Shimadzu LKB-9000) after trimethyl-silylation. The Strain had in the cell wall meso-diaminopimelic acid as a characteristic amino acid and madurose as sugars, but it did not contain arabinose, galactose or xylose.

In summary, the Strain belongs to Sugar pattern B of Cell wall type III described on page 658 of Bergey's Mannual of Determinative Bacteriology, 8th Ed., since it contains meso-diaminopimelic acid and madurose in the cell wall. The spore chains form a spiral wound closely into a doughnut, or they resemble sporangia. The color of growth is orange.about.red.about.purple, the aerial mycelium is white.about.pink, and the soluble pigment is orange.about.red--purple, and they change with pH. The growth was found to be promoted by B vitamins.

These characteristics all suggest that the Strain is a microorganism of the genus Actinomadura. Nonomura et al. identifies new five strains that belong to the genus Actinomadura (J. Ferment. Technol., Vol. 49 (11), 1971, pp. 904-912) and Actinomadura roseoviolacea is fairly close to the Strain. We therefore compared the Strain with Actinomadura roseoviolacea KCC.A-0145. The results are shown in Table 4. In spite of several differences (e.g., growth on various media and utilization of carbon sources), the two strains are similar to each other in respect of their basic characteristics. Therefore, we identified the Strain to be a new variant of Actinomadura roseoviolacea and designated it Actinomadura roseoviolacea var. biwakoensis nov. var. Actinomycetes easily undergo either artificial or natural mutation to form mutant strains, and these strains are included within Actinomadura roseoviolacea var. biwakoensis nov. var. as defined in this invention.

TABLE 4______________________________________ Actinomadura roseoviolacea Actinomadura Examination var. biwakoensis roseoviolaceaMedium Item nov. var. KCC A-0145______________________________________ISP No. 1 Growth Ring, Light Flaky growth orange.about.strong on bottom of reddish orange tube, Pale yellow Soluble Slightly, Bright None pigment reddish orangeISP No. 4 Growth Restricted, Restricted, Penetrating into Spreading, medium, Light Light reddish yellowish brown brown Aerial Scant, White Moderate, mycelium White.about.pale pinkISP No. 5V Aerial Restricted, None mycelium White.about.pale pinkISP No. 6 Soluble Slightly, None pigment Deep redWaksman No. 1V Growth Fair, Deep red Poor, Deep purple red purple Aerial Moderate, Pale None mycelium pink.about.pale yellowish pinkWaksman No. 7V Soluble None Vivid red pigment purpleUtilization of Salicin Positive Negativecarbon sources D-Sorbitol Positive Negative(using ISP (with BNo. 9) vitamins) Sucrose Positive Negative Inositol Positive Doubtful D-Mannitol Positive DoubtfulHydrolysis of Positive (weak) Negativestarch______________________________________

The production of the antibiotic A, A.sub.1, B or B.sub.1 is by fermentation, i.e., by culturing on a medium the Strain that belongs to the genus Actinomadura and which produces the antibiotic FA-1180. The Strain can be cultured by a known method of cultivating actinomycetes or modifications thereof, for example, by submerged cultivation on a liquid medium. Any type of medium can be used if it contains a nutrient source utilizable by the Strain. Any known nutrient source that is employed in the ordinary cultivation of actinomycetes can be used. Illustrative carbon sources are carbohydrates such as glycerin, glucose, molasses and starch as well as fats such as animal fat, soybean oil, cotton seed oil and peanut oil. Illustrative nitrogen sources are urea, ammonium sulfate, nitrates, meat extract, yeast extract, corn steep liquor, peptone, casein, soybean meal, cotton seed meal and peanut meal. Illustrative inorganic salts are sodium chloride, phosphate salts, potassium chloride, calcium carbonate and magnesium sulfate. Various growth promoters and defoamers may be used depending on the need. Aerobic cultivation is preferred. The cultivation temperature is generally in the range of from 20.degree. to 40.degree. C., preferably from 25.degree. to 35.degree. C., the cultivation pH is generally from 4 to 9, preferably from 5 to 7, and the cultivation condition is aerobic. Under such cultivation conditions, the production of the antibiotic A, A.sub.1, B or B.sub.1 reaches a peak within a period of 7 to 20 days, preferably 10 to 15 days, whether tank cultivation or shake cultivation is performed.

The antibiotic A, A.sub.1, B or B.sub.1 can be recovered from the culture broth by a method commonly employed in the isolation of antibiotics from the culture broth of microorganisms. Generally, the antibiotic A, A.sub.1, B and/or B.sub.1 may be recovered from the culture filtrate and/or mycelial cake by extraction with water-immiscible organic solvents. Methods that suit the physicochemical properties of the particular antibiotic are selected based on the difference in solubility in solvents, the difference in crystallizability from the solution, the difference in affinity for adsorbents and the difference in distribution between liquid phases.

The antibiotics A, A.sub.1, B or B.sub.1 thus obtained has the physicochemical properties set forth below.

A. Properties of FA-1180 A

(1) Color and nature: amphoteric dark red, needle-shaped crystal

(2) Molecular weight: 659 (FD-Mass)

(3) Elemental analysis: Found: C 60.9%, H 6.2%, N 2.4%

(4) Melting point: 186.degree. to 188.degree. C. (with decomposition) (powder) 155.degree. to 160.degree. C. (with decomposition) (crystal)

(5) IR spectrum (determined by KBr method, see FIG. 7 which is an IR spectrum of antibiotic FA-1180 A as determined by KBr method): peaks at 3435-3310, 2920, 1708, 1586, 1388, 1292, 1230, 1198, 1168, 1110, 1008 and 968 cm.sup.-1

(6) UV and visible spectrum (determined in methanol sol., see FIG. 11 which is a UV and visible spectrum of antibiotic FA-118 A, wherein the solid line is an absorption curve obtained in methanol solution, the broken line is obtained in 0.01 N hydrochloric acid/methanol solution, and the long and short dash line is in 0.01 N sodium hydroxide/methanol solution): 237 m.mu. (E.sub.1 cm.sup.1% 980), 255 m.mu. (E.sub.1 cm.sup.1% 760), 292 m.mu. (E.sub.1 cm.sup.1% 230), 493 m.mu. (E.sub.1 cm.sup.1% 390), 530 m.mu. (E.sub.1 cm.sup.1% 270)

(7) TLC chromatography: TLC on silica gel with a developing solvent (chloroform:toluene:methanol=7:3:3); Rf=0.45

(8) Solubility: freely soluble in methanol, soluble in chloroform, slightly soluble in n-hexane

(9) PMR spectrum: see FIG. 15 which is a PMR spectrum of antibiotic FA-1180 A (60 MHz, in CDCl.sub.3)

(10) Specific rotation: [.alpha.].sub.D.sup.24 +147.degree. (c=0.011, in CHCl.sub.3)

B. Properties of FA-1180 A.sub.1

(1) Color and nature: amphoteric dark red needle-shaped crystal

(2) Elemental analysis: Found: C 58.7%, H 5.6%, N 1.9%

(3) Melting point: 175.degree. to 185.degree. C. (with decomposition) (powder)

(4) IR spectrum (see FIG. 8 which is an IR spectrum of antibiotic FA-1180 A.sub.1 as determined by KBr method): peaks at 3435-3310, 2920, 1708, 1586, 1388, 1292, 1230, 1198, 1168, 1110, 1008 and 968 cm.sup.-1

(5) UV and visible spectrum (determined in methanol sol., see FIG. 12 which is a UV and visible spectrum of antibiotic FA-1180 A.sub.1, wherein the solid line is an absorption curve obtained in methanol solution, the broken line is obtained in 0.01 N hydrochloric acid/methanol solution, and the long and short dash line is in 0.01 N sodium hydroxide/methanol solution): 234 m.mu. (E.sub.1 cm.sup.1% 457), 254 m.mu. (E.sub.1 cm.sup.1% 351), 292 m.mu. (E.sub.1 cm.sup.1% 101), 492 m.mu. (E.sub.1 cm.sup.1% 172), 527 m.mu. (E.sub.1 cm.sup.1% 132), 580 m.mu. (E.sub.1 cm.sup.1% 30)

(6) TLC chromatography: TLC on silica gel with a developing solvent (chloroform:toluene:methanol=7:3:3); Rf=0.50

(7) Specific rotation: [.alpha.].sub.D.sup.24 +185.degree. (c=0.0085, in CHCl.sub.3)

C. Properties of FA-1180 B

(1) Color and nature: amphoteric dark red needle-shaped crystal

(2) Elemental analysis: Found: C 59.9%, H 5.4%, N 2.3%

(3) Melting point: 198.degree.-215.degree. C. (with decomposition) (powder) 144.degree.-146.degree. C. (with decomposition) (crystal)

(4) IR spectrum (determined by KBr method, see FIG. 9 which is an IR spectrum of antibiotic FA-1180 B as determined by KBr method): peaks at 3405, 2960, 1586, 1543, 1410, 1388, 1292, 1238, 1193, 1110, 1008 and 968 cm.sup.-1

(5) UV and visible spectrum (determined in methanol sol., see FIG. 13 which is a UV and visible spectrum of antibiotic FA-1180 B, wherein the solid line is an absorption curve obtained in methanol solution, the broken line is obtained in 0.01 N hydrochloric acid/methanol solution, and the long and short dash line is in 0.01 N sodium hydroxide/methanol solution): 237 m.mu. (E.sub.1 cm.sup.1% 850), 255 m.mu. (E.sub.1 cm.sup.1% 660), 295 m.mu. (E.sub.1 cm.sup.1% 200), 492 m.mu. (E.sub.1 cm.sup.1% 350), 528 m.mu. (E.sub.1 cm.sup.1% 250)

(6) TLC chromatography: TLC on silica gel with a developing solvent (chloroform:toluene:methanol=7:3:3); Rf=0.30

(7) Solubility: freely soluble in methanol, soluble in chloroform, slightly soluble in n-hexane

(8) Specific rotation: unfixed [.alpha.].sub.D.sup.24 +88.degree..about.-70.degree. (c=0.01075, in methanol:chloroform=1:9)

D. Properties of FA-1180 B.sub.1

(1) Color and nature: amphoteric dark red needle-shaped crystal

(2) Elemental analysis: Found: C 58.4%, H 7.3%, N 19%

(3) Melting point: 152.degree. to 154.degree. C. (with decomposition) (crystal)

(4) IR spectrum (see FIG. 10 which is an IR spectrum of antibiotic FA-1180 B.sub.1 as determined by KBr method): peaks at 3420, 2960, 2920, 1600, 1420, 1300, 1230, 1200, 1160, 1120, 1060, 1010, 960 and 820 cm.sup.-1

(5) UV and visible spectrum (determined in methanol sol., see FIG. 14 which is a UV and visible spectrum of antibiotic FA-1180 B.sub.1, wherein the solid line is an absorption curve obtained in methanol solution, the broken line is obtained in 0.01 N hydrochloric acid/methanol solution, and the long and short dash line is in 0.01 N sodium hydroxide/methanol solution): 234 m.mu. (E.sub.1 cm.sup.1% 603), 253 m.mu. (E.sub.1 cm.sup.1% 487), 293 m.mu. (E.sub.1 cm.sup.1% 133), 491 m.mu. (E.sub.1 cm.sup.1% 249), 525 m.mu. (E.sub.1 cm.sup.1% 186)

(6) TLC on silica gel with a developing solvent (chloroform:toluene:methanol=7:3:3); Rf=0.45

(7) Specific rotation: unfixed [.alpha.].sub.D.sup.24 +165.degree..about.+96.degree. (c=0.013, in methanol:chloroform=1:9)

The present inventors supposed chemical structures of the present antibiotic A, A.sub.1, B or B.sub.1 from results of various measurements, consequently the present antibiotic is identified as anthracycline glycosides having the formula (I): ##STR2##

When R in the formula (I) represents an acetyl group, the present antibiotic is FA-1180 A or A.sub.1. When R represents a 1-hydroxyethyl group, the present antibiotic is FA-1180 B or B.sub.1. The present antibiotic A and A.sub.1 or the present antibiotic B and B.sub.1 are a stereoisomer, respectively. A carbon-13 nuclear magnetic resonance spectrum of the antibiotic was determined with an FX-60 Spectorometer (JEOL) operating at 15.04 MHz by dissolving about 30 mg of the antibiotic in 0.5 ml of CDCl.sub.3 or in CD.sub.3 OD-CDCl.sub.3 mixture and using tetramethylsilane (TMS) as the internal reference. Observed chemical shifts (in ppm relative to tetramethylsilane) and multiplicities for .sup.13 C magnetic resonance spectra of the antibiotic are as follows:

__________________________________________________________________________The Antibiotic .sup.13 CMR Listings Assign- No. Carbon*ofments plicityMulti- A(CDCl.sub.3) A.sub.1 (CDCl.sub.3) ##STR3## ##STR4##__________________________________________________________________________1 13 s or d 211.7 211.7 73.6 73.22 12 s 190.0 190.3 190.3 190.23 5 s 185.7 185.9 186.1 186.04 4 s 162.4 162.4 162.4 162.45 6 s 156.6 156.8 157.2 157.26 11 s 156.5 156.5 156.7 156.77 2 d 136.9 136.9 136.9 136.98 12a s 136.6 136.8 138.0 138.19 6a s 133.9 133.9 134.8 134.810 10a s 133.0 133.3 133.4 133.311 3 d 124.8 124.7 124.7 124.612 1 d 119.4 119.6 119.5 119.513 4a s 115.8 115.8 116.0 115.914 5a s 110.0 111.1 111.1 110.915 11a s 110.2 110.3 110.2 110.116 9 s 76.4 76.6 71.9 71.817 7 d 69.2 69.5 70.1 70.218 10 t 34.4 36.2 34.3 36.019 8 t 33.8 34.9 34.2 34.020 14 q 24.8 24.8 (16.8) (16.7)21 1' d 100.5 101.1 100.8 100.922 2' t 32.9 33.6 32.9 32.923 3' d 46.0 45.7 46.1 45.624 4' d 74.7 82.0 76.0 81.325 5' d 63.8 64.2 64.0 64.426 6' q 16.3 17.0 (16.6) (16.7)27 1" d 101.0 106.7 101.7 106.228 2" t 42.3 45.4 42.9 45.829 3" d 72.7 75.8 73.3 75.930 4" q 23.7 23.3 23.4 23.131 5" d 67.5 67.9 67.8 67.832 6" t 66.2 66.8 66.5 66.533 7" q 17.3 18.1 (17.4) (18.0)__________________________________________________________________________ *Note: Assignments of carbon are shown in aforementioned formula (I).

The antibiotic A, A.sub.1, B or B.sub.1 is capable of reacting with a variety of acidic or basic substances to form the corresponding and pharmaceutically acceptable salts. A variety of acidic substances are an organic acid such as acetic acid, propionic acid, etc., a mineral acid such as hydrochloric acid, sulfuric acid, etc., and the others. A variety of basic substances are a hydroxide or carbonate of an alkali metal such as sodium, potassium, etc., and the others.

TEST EXAMPLE 1

The minimum inhibitory concentration (MIC) of the FA-118O A, A.sub.1, B or B.sub.1 was determined by the agar dilution method, and the results are indicated in Table 5 below.

TABLE 5______________________________________ MIC (ppm) FA- FA- FA- FA-Microorganisms 1180 A 1180 A.sub.1 1180 B 1180 B.sub.1______________________________________Bacillus subtilis PCI 219 1.56 1.56 6.25 3.125Bacillus subtilis 17 A 1.56 1.56 12.5 12.5Bacillus subtilis 45 T <0.2 <0.2 0.4 0.4Bacillus cereus IAM 1729 3.125 -- 25 --Staphylococcusaureus 209 P 12.5 12.5 50 50Staphylococcus aureus 12.5 12.5 50 50Escherichia coli NIHJ >100 >100 >100 >100Escherichia coli W3110 >100 >100 >100 >100Escherichia coli P3478 >100 >100 >100 >100Pseudomonas aeruginosa H >100 >100 >100 >100Salmonellatyphimurium IFO 12529 >100 >100 >100 >100Klebsiellapneumoniae IFO 3512 >100 >100 >100 >100Serratiamarcescens IFO 12648 >100 >100 >100 >100Candida albicans >100 >100 >100 >100______________________________________ Notes: (1) Medium: Brain heart infusion agar (2) Determined: 18 hrs after inoculation

TEST EXAMPLE 2

Table 6 shows the results of a test for determining the therapeutic effect of the FA-1180 A, B and B.sub.1 against P-388 Mice Leukemia. The test was conducted in accordance with the method described in Cancer Chemotherapy Reports, Part 3, Vol. 3, No. 2, p. 9, September 1972. Mice inoculated intraperitoneally with 1.times.10.sup.6 cells of P-388 were administered with the test compounds daily on days 1 to 5. The activity (T/C) of the test compounds is represented by the ratio of the median survival time of the treated group to that of the control group.

TABLE 6______________________________________Antibiotic Dose (mg/kg/day) Activity (T/C) (%)______________________________________FA-1180 A 1.0 205 0.5 167 0.25 148FA-1180 B 1.0 190 0.5 162 0.25 143FA-1180 B.sub.1 1.0 179 0.5 168 0.25 151Control: 5-FU 20 198______________________________________

TEST EXAMPLE 3

The tumor cell suspension was prepared from cells taken from ascites of rats to which 1.times.10.sup.6 cells of Yoshida rat sarcoma had been inoculated before 3 to 5 days. The ascites was diluted with a medium which was constituted from 80 to 90% (v/v) Fischer's solution containing 20 to 10% (v/v) desensitized horse serum. One half milliliter of the cell suspension containing 5.about.20.times.10.sup.4 cells thus prepared was placed in a small test tube. A substance to be tested was dissolved and diluted with the same medium and 0.5 ml of the diluted solution was added to the cell suspension. To the control tube was added 0.5 ml of the medium. 2 to 5 tubes were used for each experimental units. They were incubated statically at 37.degree. C. for 2 to 3 days. After the incubation, growth was measured by observing the quantity of precipitated cells on the bottom of the test tube with the naked eyes. Growth inhibitory effect of a substance was expressed by the concentration pointing 50 percent growth of the control growth.

TABLE 7______________________________________ Growth Inhibitory Effect IC.sub.50Antibiotics (.mu.g/ml)______________________________________Antibiotic FA-1180 A 10.sup.-2Antibiotic FA-1180 A.sub.1 10.sup.-3Antibiotic FA-1180 B 10.sup.-2Antibiotic FA-1180 B.sub.1 10.sup.-2Adriamycin hydrochloride 10.sup.-2______________________________________ IC.sub.50 : The concentration pointing 50% growth of the control growth.

The antibiotics A, A.sub.1, B and B.sub.1 are classified as anthracycline glycosides, but the antibiotics of this invention had novel chemical structure and none of the documented anthracycline glycosides had the same physicochemical properties as those of the antibiotics of this invention. Therefore, we concluded that the antibiotics FA-1180 A, A.sub.1, B and B.sub.1 and salts thereof are novel substances.

The antibiotics A, A.sub.1, B and B.sub.1 and salts thereof have a strong ability to inhibit the growth of Gram-positive bacteria and they also have antitumor activity, so they are useful as an active ingredient for medicines such as disinfectants for medical devices or chemotherapeutics against microorganisms in human beings and animals. Especially the antibiotic B is useful as an active ingredient for medicines. The antibiotics of this invention are formulated and used in a manner analogous to that for ordinary antibiotics for medical applications. For example, the antibiotics of the invention and an inert diluent are formulated in a powder, tablet, lozenge, capsule, suspension, syrup, cream, ointment or injection, and used. Usually, these antibiotics are administered at a dosage of 0.1 to 20 mg/kg/day.

This invention is now described in greater detail by reference to the following example which is given here for illustrative purposes only and is by no means intended to limit the scope of the invention.

EXAMPLE

(1) Fermentation

One liter of a broth containing 20 g of glucose, 10 g of corn steep liquor, 10 g of soybean meal, 2 g of potassium nitrate, 5 g of sodium chloride and 0.5 g of magnesium sulfate and which was adjusted to a pH of 7.0 was prepared. Fifteen milliliters of the broth was placed in a 50 ml test tube. The tube was sterilized by the conventional method. The broth in the test tube was inoculated with spores of the strain FA-1180 which were subjected to a shaking cultivation at 30.degree. C. for 10 days to produce a first seed. A second seed was prepared in a similar manner by shaking cultivation at 30.degree.-35.degree. C. in a 2-liter flask containing 350 ml of the remaining broth. Then, a 50-liter jar fermentor was charged with 35 liters of a broth having the same composition as specified above, and after sterilization, the broth was inoculated with 700 ml of the second seed which was cultured at 28.degree.-30.degree. C. for 14 days with air supplied at a rate of 35 liters per minute and the agitator rotated at 250 rpm.

(2) Extraction

After the fermentation, 70 liters of the liquid culture was separated into the mycelial cake and filtrate by filtration. The cake was extracted three times with 3-liter portions of acetone, followed by concentration under vacuum. The concentrate was extracted twice each with a mixture comprising 300 ml of water, 200 ml of a saturated aqueous solution of sodium bicarbonate and one liter of chloroform. After concentrating the chloroform layer in vacuum, the concentrate was extracted three times with 500 ml portions of aqueous solution of 0.01 N acetic acid. The resulting orange extract was neutralized with an aqueous solution of 2 N sodium hydroxide until it turned dark red, and the extract was again extracted with one liter of chloroform. The extract was then concentrated in vacuum, mixed with a great excess of n-hexane to form a precipitate which was filtered off and dried to give about 200 mg of a dark red crude powder.

(3) Purification

(3-1) A solution of 200 mg of the crude powder in a small amount of methanol was applied to twenty silica gel sheets for TLC chromatography (Art. 5553 of Merck & Co., Inc.) and developed to 15 cm from the placing point with a solvent comprising chloroform:toluene:methanol=7:3:3. After the development, red spots having Rf values of 0.45 and 0.3 were collected and extracted with methanol. The respective extracts soluble in chloroform were concentrated to give 28 mg of FA-1180 A and 7 mg of FA-1180 B.

(3-2) Further purification of the complex (160 mg) into its components was achieved by droplet countercurrent chromatography (DCC) with chloroform:methanol:0.1 N acetic acid=5:5:3 as solvent system. DCC apparatus contained 300 separation tubes was used and separation was accomplished by down flow method. The effluent was monitored by TLC on silica gel plate with a solvent system of chloroform:toluene:methanol (7:3:3) or lower phase of chloroform:methanol:0.1 N acetic acid (5:5:3).

The fractions containing each components were pooled separately and adjusted to pH 8.6 with sodium bicarbonate saturated solution. In this treatment, the each fraction was separated into a solvent layer and an aqueous layer. Separated solvent layer washed with deionized water and dried with anhydrous sodium sulfate, concentrated in vacuo to small volume, and then crystallized from chloroform-n-hexane for FA-1180 A (40 mg) and FA-1180 A.sub.1 (8 mg) or chloroform for FA-1180 B (7 mg) and FA-1180 B.sub.1 (7 mg).

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

1. An anthracycline antibiotic selected from the group consisting of FA-1180 B and FA-1180 B.sub.1, or a pharmaceutically acceptable salt thereof, said antibiotic having the formula (I): ##STR5## wherein R represents a 1-hydroxyethyl group.